1. Field of the Invention
The present invention relates to a method for rapidly screening gene amplification products in commercially available polypropylene microtiter plates.
2. Background of the Art
Gene amplification reactions are powerful tools for generating multiple copies of a nucleic acid sequence from a few. A variety of methods are available, such as, polymerase chain reaction (PCR), self-sustained sequence replication (3SR), beta-Q replicase (.beta.QR), first strand synthesis with DNA polymerase, ligation amplification reaction or ligase-based amplification system (LAR/LAS) and reverse transcription.
Polymerase chain reaction (PCR) is a powerful method for amplifying DNA, and is used in a variety of research fields in both basic biosciences and clinical research. Saiki, R. et al. Science, 230:1350-1354 (1985). PCR has been labeled as one of the most promising techniques for the diagnosis and/or analysis of various disease-related genes. Bernstam, V. A. in "Handbook of Gene Level Diagnostics in Clinical Practice", pp. 19-69 (CRC Press, Boca Raton, Fla., 1992). However, manipulating large numbers of test samples simultaneously has presented an important technical obstacle.
Following gene amplification, test samples are typically analyzed to quantify the amount of the nucleic acid produced. Ordinarily, three well known analytical techniques are used: electrophoresis; hybridization blots; or spectrophotometry.
In electrophoresis, agarose or acrylamide gels containing the amplified fragments are run, followed by staining with an intercalating agent such as ethidium bromide. In hybridization blots, such as Southern blots and Dot blots, the samples are analyzed by annealing with specifically labeled probes. Bernstam, V. A. in "Handbook of Gene Level Diagnostics in Clinical Practice", pp. 19-69 (CRC Press, Boca Raton, Fla., 1992). Both of these analytical techniques are time-consuming and cumbersome when large numbers of test samples are generated.
In the third technique, spectrophotometry, the amount of DNA in a sample is determined by measuring the optical density (OD) of the sample at 260 nm. Sambrook, J., Fritsch, E. F. & Maniatis, T. in "Molecular Cloning, A Laboratory Manual" (2d ed): pp. E.5. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989). Alternatively, the sample can be labeled with a DNA-binding fluorescent dye and the fluorescence of the sample measured at the dye's excitation wavelength. For example, Hoechst 33258 dye has been used with some success. In one example, a sample was labeled with proprietary dyes and read at a wavelength of 345 nm. Mocharla, R., Mocharla, H. & Hodes, M. E. Nucleic Acids Res.:, 15:10589 (1987). However, measurements of polynucleotide concentrations at OD.sub.260 or the optimal excitation wavelength for Hoechst dye (345 nm) are not very sensitive through polystyrene or polyvinyl chloride materials. It would provide a tremendous advantage to have gene amplification reactions performed in microtiter wells that were more sensitive to spectrophotometric measurments.
There are a variety of commercially available fluorescent dyes, such as, thiazole orange, or dyes from the general groups of benzoxazolium-4-pyridinium, benzothiazolium-4-pyridinium, benzoxazolium-4-quinolinium, and benzothiazolium-4-quinolinium florescent dyes that show good spectrographic sensitivity for the analysis of nucleic acid sequences. These dyes have excitation wavelengths in the low 400 nm range and emission wavelengths in the high 600 nm range when bound to a nucleic acid sequence. Such dyes are available from Molecular Probes, Inc.
A relatively new dye is Yoyo-1, which has shown promising spectrographic sensitivity for detecting nucleic acids. Glazer, A. N., Rye H. S. Nature, 359:859-861 (1992). Yoyo-1 has an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
Conventionally, microtiter plates have been made from heat sensitive polymeric materials, such as polystyrene and polyvinyl carbonate. These plates do not easily tolerate the repeated heat denaturing processes during PCR. Recently, Coster Corporation (Cambridge, Mass.) and Nunc Corporation (Naperville, Ill.) introduced microtiter plates made from polypropylene. Such plates are significantly more heat stable than polystyrene plates.
However, polypropylene is a partially opaque material, i.e., it appears cloudy or not fully transparent. Thus, it is difficult to see samples once they are in the wells.
Currently, some manufactures provide PCR equipment that could be suitable for analysis of microtiter plates (Integrated Separation Systems, Natick, Mass.; MJ Research, Watertown, Mass., Techne, Princeton, N.J., etc.). Therefore, PCR could, in theory, be easily carried out in microtiter plates.
Accordingly, it would be a great advantage if a system was developed for conducting gene amplification reactions in microtiter plates where both the reaction and the analysis could take place in a single plate without the need for transfer of the products.